Ubiquitin is one of the cell's most broadly deployed covalent modifiers of proteins. Protein ubiquitination serves critical regulatory roles in virtually every aspect of cell and developmental biology, and we've come to appreciate that particular ubiquitination misevents underlie many debilitating and deadly human diseases including cancer and neurodegenerative disorders. Ubiquitination occurs through an enzymatic cascade in which ubiquitin is passed from a ubiquitin activase to a ubiquitin conjugase, and then attached to a substrate by a ubiquitin ligase, which imparts the substrate specificity. Because we know little about the substrate cohort of most ubiquitin ligases, functions for the nearly 1000 predicted human ubiquitin ligases have not been characterized. No routine technologies currently exist to identify substrates of ubiquitin ligases. This hampers both the advancement of the ubiquitination field as well as fields in which ubiquitination serves a key role, such as stem cell biology, cancer, and neurodegenerative disorders. We propose to address this overarching cell biological problem by developing mass spectrometry- based ubiquitin proteomics methods to identify substrates for ubiquitin ligases. To develop our methodology, we will take advantage of the genetic and biochemical tractability of yeast, a simple eukaryote. Because ubiquitination is conserved in all eukaryotes, most yeast ubiquitination pathways have human counterparts. Consequently, we will learn valuable biological information while providing a template for the development of a mammalian version of this technology. The success of our biological objective is highly dependent on computational proteomics. We therefore propose a coordinated effort between ubiquitin biologists and computational scientists to accomplish the following aims: (1) Improve experimental design and develop statistical methods to identify putative substrates of ubiquitin ligases by tandem mass spectrometry (MS/MS);(2) Develop refined experimental protocols to reduce the complexity of the ubiquitin proteome samples;(3) Expand the statistical analysis of the mass spectrometry data to exploit a hybrid approach using MS/MS and LC-MS data. PUBLIC HEALTH RELEVANCE: The modification of cellular proteins by the ubiquitin is one of the cell's most important means to tightly control protein function. Protein ubiquitination is so broadly used that the human cell possesses nearly 1000 ubiquitin ligases to add this critical modification to proteins, but we know very little about the cohort of substrates for any one of these enzymes. We will develop cutting-edge experimental and computational technologies to identify the substrate cohorts for any desired ubiquitin ligase, especially those implicated in human diseases such as cancer and neurodegenerative disorders.